Electrophoresis is one of the most widely used separation techniques in the biologically-related sciences. Molecular species such as peptides, proteins, and oligonucleotides are separated by causing these species to migrate in a buffer solution under the influence of an electric field. This buffer solution normally is used in conjunction with a low-to-moderate concentration of an appropriate gelling agent such as, for example, agarose or polyacrylamide, to minimize the occurrence of mixing of the species being separated. Two primary separating mechanisms exist: a) separations based on differences in the effective charge of the analytes; and b) separations based on molecular size.
The first of these mechanisms is generally limited to low or moderate molecular weight materials, such as, for example, oligonucleotides. This is because the effective charges of high molecular weight materials become rather similar, making it difficult or impossible to separate them.
Separations based on molecular size are generally referred to as molecular "sieving". Molecular sieving relies upon gel matrices having controlled pore sizes as the separating medium. In such separating systems, if the effective charge of the analytes are the same, the separation results from differences in the abilities of the different size molecular species to penetrate through the gel matrix. Smaller molecules move relatively more quickly than larger ones through a gel of a give pore size.
Oligonucleotides and medium-to-high molecular weight polypeptides and proteins are commonly separated by molecular sieving electrophoresis, although in the case of proteins, charge and size can be used independently to achieve separations. In the case of proteinaceous materials, however, it is generally necessary to modify the materials to be separated so that they all have the same effective charges. This is commonly accomplished by employing an SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) derivitization procedure, such as discussed in Gel Electrophoresis of Proteins: a Practical Approach (2nd edition, B.D. Hames & D. Rickwood, Eds., IRL Press, Oxford University Press, 1990. See also, New Directions in Electrophoretic Methods, J.W. Jorgenson Ampersand M. Phillips, Eds., Published by American Chemical Society, Washington, D.C., 1987. Both of these references are incorporated herein by reference.
Occasionally, it is desirable to separate proteinaceous materials under conditions which pose a minimal risk of denaturation of the protein. In such cases, some additives such as urea and SDS, which can cause denaturation of the protein, are avoided. Because of this, the resulting separations are predicated upon differences in both the molecular size and charge of the constituents of the materials.
Most electrophoretic separations are presently conducted in slabs or open beds. These separations are very difficult to automate or quantitate. Extremely high resolution separations of materials having different effective charges has been achieved by open tube free zone electrophoresis and isotachophoresis in narrow capillary tubes. In addition, bulk flow (i.e., the flow through the capillary tube of the electrophoresis buffer and the sample being analyzed) can be driven by electroosmosis to yield very sharp electropherograms peaks. Such high efficiency, open tube electrophoresis has not generally been applied to the separation of medium-to-high molecular weight oligonucleotides, however, because these materials have very similar effective charges, as previously noted. Furthermore, open-tube electrophoresis does not provide size selectivity for proteinaceous materials. Achieving high resolution separations of, e.g., oligonucleotides, is directly related to the gel containing microcapillaries utilized in the electrophoresis system. Accordingly, and because of the importance of high performance capillary electrophoresis as a separating technique in the biological sciences, significant attention has been paid to the gel-materials which are utilized in this technique.
Polymeric gel materials typically employed in high performance capillary electrophoresis are usually any crosslinked polymer which has a pore structure which can be adjusted by varying the amounts of monomer and crosslinking agent, as well as the reaction conditions. Preferred polymeric gel materials are based on acrylamide and N'-methylenebisacrylamide, N'-methylenebisacrylamide serving as a cross linking agent. Other crosslinking agents include N,N'-(1,2-dihydroxyethylene)-bisacrylamide, N,N'-diallyltartardiamide, N,N'-cystamine-bisacrylamide, and N-acryloyltris(hydroxymethyl) aminomethane. Gel polymerization is typically initiated by ammonium persulfate and N,N,N', N'-tetramethylethylenediamine (TEMED).
The pore size of polyacrylamide gels is dependent on total monomer concentration (% T) and on the concentration of crosslinker (% C). Pore size can be progressively increased by reducing % T at a fixed % C; however, very dilute gels are mechanically unstable and pore sizes greater than 80nm cannot be obtained. The alternative approach is to progressively increase % C at fixed % T where the increase in pore size is considered to be due to the formation of a bead-like structure rather than a 3-D lattice. In this procedure, stable gels of high pore size (about 200-250nm) can be obtained at 30% C N'-methylenebisacrylamide; however, at higher concentrations, the gels become hydrophobic and prone to collapse.
While the capillary gels themselves have been rather well defined and analyzed in the art, preparation of the capillary tubes including such gels has not been well defined. For example, conventional gel-containing microcapillary columns maintain the capillary gel within the column by way of covalent attachment of the gel to the column. See, for example, S. Hjerte, "High-Performance Electrophoresis Elimination of Electroosmosis and Solute Absorption." J. Chrom., 347:191-198(1985); U.S. Pat. No. 4,865,707 (Karger et al) and U.S. Pat. No. 4,997,537 (Karger et al). The foregoing references are incorporated herein by reference. The gel-containing microcapillary columns described in the preceeding references suffer from at least the following problems: lot to lot reproducibility is unpredictable; the stability of the columns themselves is subject to failure after approximately 50-150 capillary runs; and the reaction time necessary for preparation of such columns can take as long as about 24 hours.
While capillary electrophoresis technology is generally directed towards the research environment, it has recently been suggested that this analytical technique has the potential for use as a clinical investigative technique. See "Capillary Electrophoresis: Tool for Clinical Diagnosis?", Clin. Lab Letter 12, 4: 1-2 (1991). While it is important in a research environment to ensure, e.g., lot to lot reproducibility and stability of the column, these concerns would be exacerbated in a clinical setting where patient analytical results require consistency from the analytical techniques utilized. Improved gel-filled capillary columns for electrophoresis which provides superior stability greater lot-to-lot reproducibility, improved resolution and shorter reaction times would be of great value to those fields that utilize such an analytical technique.